The technical field of the present invention is that of biological analysis. More particularly, the present invention relates firstly to a device for lysis of microorganisms present in an environmental or clinical sample, for extraction and for purification of the nucleic acids of said microorganisms. The invention further relates to an automated system for lysis of the microorganisms, for extraction and for purification of the nucleic acids of said microorganisms, for purposes of analysis.
An upsurge in nosocomial infections in hospitals has been observed for several years. These infections are explained by contamination of hospitalized, and therefore by definition immunodepressed, persons by pathogenic microorganisms present in the hospital environment and not destroyed despite considerable care always being applied to the disinfection of equipment and surfaces and to air treatment. In view of these more and more frequent cases of environmental microbiological contamination, the development of devices and methods for improving and facilitating environmental controls has become a major challenge for health professionals.
In addition to the problem of nosocomial infections, control of environmental conditions has also been a recurrent concern in industry for many years, in particular in food industries, and pharmaceutical or cosmetic industries. In food industries, the disastrous consequences for consumers' health that can arise from contamination of products, or even of raw materials, by a pathogenic microorganism are well known. In fact, food poisoning due to bacteria such as those of the genus Listeria or Salmonella is now a common occurrence. Control of air quality is also a key process in the quality approach of pharmaceutical or cosmetic industries.
Moreover, these controls must comply with ever increasing requirements, as regulations become stricter all the time.
Among the tools at the disposal of health professionals or manufacturers for carrying out environmental controls, microbiological air samplers are solutions of choice for the detection of airborne microorganisms. These devices are placed at suitable points in places where measurement of microbiological air contamination is required. They are generally constituted of an air sampler coupled to a culture medium. The air collected by the air sampler comes into contact with the culture medium; any microorganisms contained in the air collected will be deposited on the culture medium. The culture medium is then recovered and placed in a stove to promote growth of the microorganisms. It is thus possible to detect and identify said microorganisms by conventional microbiological techniques.
These devices nevertheless have a major drawback, which is connected with the technology used. This drawback is the time taken to obtain the analysis result. In fact, the use of conventional techniques of microbiology, in particular of bacteriology, requires incubation times necessary for cell growth, or even phases of re-seeding on specific culture media to permit identification. As a result, the time taken to obtain a result is relatively long, or even too long, when we are trying to detect and identify a pathogen that is responsible for a nosocomial infection or for food poisoning.
Another drawback of a device of this type is that although the use of culture media makes it possible to discriminate between genera and species of bacteria, generally it does not allow discrimination of the strains of one and the same bacterial species. Now, it is known that the pathogenicity of a microorganism can vary significantly depending on the strain in question.
Moreover, this type of device has the drawback that it is unable to detect airborne microorganisms that are viable but cannot be cultured.
There are, moreover, devices intended for recovery of airborne particles, in particular microorganisms. Thus, document GB-2 254 024 describes a device for collecting airborne particles whose principle is based on the cyclone effect. Although such a device is found to be suitable for collecting airborne particles, including microorganisms, it has never been investigated for treating the sample thus obtained, in particular for extracting genetic material intended to be used for analysis.
More generally, the techniques that are most relevant in terms of identification of microorganisms and/or of speed of delivering the results, whether with respect to clinical or environmental samples, are undoubtedly the techniques of molecular diagnostics. These techniques, based on analysis of the genetic material of the microorganisms, and in particular of certain specific sequences of interest, make it possible to obtain a very precise identification of microorganisms in record time, since they make it possible to omit the culture steps.
Nevertheless, the use of such techniques has certain limits, the most important of which is the potentially limited quantity of microorganisms that are present in the air and therefore recoverable for performing the analysis. In fact, it is known that environmental samples, as well as certain clinical samples, are relatively poor in microorganisms. Consequently, only a small amount of genetic material is obtained from this raw material. The performance of the technique used for extracting the nucleic acids, in terms of yield, then becomes a crucial parameter.
Moreover, most of the existing techniques for lysis of microorganisms are not of general application for all microorganisms and/or require the intervention of qualified personnel for taking care of the manual stages.
Document WO-A-2005/038025 describes a method for extracting nucleic acids from microorganisms, notably from air samples. This method comprises the use of three different methods of lysis, namely chemical lysis, lysis by thermal shock and mechanical lysis. Although such a method undoubtedly makes it possible to optimize the efficiency of extraction of the nucleic acids and therefore to increase the amount of genetic material available for analysis, this efficiency still depends on the quantity of microorganisms recovered. Yet, nothing is described in that document for optimizing the recovery of said microorganisms.
Document U.S. Pat. No. 5,707,861 describes a device for disrupting living cells such as microorganisms. This device makes it possible to lyse the cells by utilizing glass beads, but in addition by the effect of vibration due to the gap that exists between the tubes containing the microorganisms and the holes in the support holding said tubes. Thus, a device of this kind makes it possible to optimize cell lysis and therefore optimize extraction of the genetic material. Said device and the method employed by the latter have the same limitations as those mentioned above, namely they still depend on the quantity of microorganisms recovered. Moreover, they have the additional drawback of requiring a subsequent stage of concentration of the nucleic acids in order to isolate them from the cellular debris. Finally, they require manual recovery of the nucleic acids, at the end of the concentration stage.
These problems also arise with the device described in document U.S. Pat. No. 5,567,050.
Systems that are more integrated have also been described. Thus, document WO-A-2004/018704 describes a device, and an associated method using the PCR (Polymerase Chain Reaction) amplification technique, for collecting microorganisms from the air and identifying them. This system is particularly suitable for combating attempted attacks by biological contamination in postal sorting centers. This system is composed of an air sampler positioned along the circuit for transporting the mail, a device for filtration/separation of particles by the cyclone effect, a device for concentration/recovery of the particles in a liquid sample, and a device for transferring a fraction of the sample to a GeneXpert™ PCR analysis cartridge from the company Cepheid. The cartridge is then transferred manually to a separate automatic biological analyzer for identification of the microorganism or microorganisms collected from the air.
Although this system makes it possible to solve a great many of the technical problems associated with the devices and methods described above, it nevertheless has some major drawbacks. The first of these drawbacks is that the system for treatment of the sample (collection, separation, concentration/recovery) prior to transfer to the analysis cartridge is relatively complex and expensive. Another drawback is that there is no GeneXpert™ cartridge that can perform both lysis and purification of nucleic acids. Yet a second drawback is that the microorganisms collected are recovered in a liquid sample, only a fraction of which is analyzed. This means that the risk of not recovering all of the microorganisms and therefore all of the nucleic acids is very high, greatly restricting the relevance of the analysis. Moreover, despite its complexity, this system requires manual transfer of the cartridge to the GeneXpert™ automatic analyzer.
Thus, a first aim of the present invention is to provide a device, a system and a universal method of lysis, which are effective both for environmental and clinical samples, for a great variety of microorganisms, whether they are bacteria, viruses or fungi, optionally in the vegetative state or in the form of spores.
Another aim of the present invention is to provide a device suitable for efficiently lysing said microorganisms contained in an environmental sample such as air, or in a clinical sample, in order to extract the nucleic acids from it and recover said nucleic acids for the purpose of analysis, in an integrated manner.
Another aim of the present invention is to provide a device suitable for collecting all of the microorganisms contained in an air sample.
Another aim of the present invention is to provide a device of simple design, minimizing the number and the complexity of the manual stages.
Another aim of the present invention is to provide a device that is extremely compact.
Another aim of the present invention is to provide a closed device in which the various stages enumerated above take place without risk of external contamination.
Another aim of the present invention is to provide a device in which said stages take place without transfer of the sample by the operator, thus preventing contamination of the latter.
Another aim of the present invention is to provide a device and a system that limits human intervention and improves the traceability of sample treatment.
Finally, another aim of the present invention is to provide a device and an automated system capable of supplying target nucleic acids in a buffer that can be used directly in stages of molecular diagnostics comprising for example stages of amplification and of detection, without additional stages of pretreatment such as centrifugation or filtration.